Alex Brown - US grants

Parathyroid glands (PTGs) express a calcium-sensing receptor (CaR) that detects the concentration of extracellular calcium and signals the glands to secrete the proper amount of parathyroid hormone (PTH) to maintain normal calcium levels. The calcium-PTH relationship is sigmoidal and can be defined by four parameters: maximum PTH, minimum PTH, the midpoint of the curve (set-point), and the slope at the midpoint. The CaR levels are decreased in the hyperplastic PTGs of patients with secondary HPT (2oHPT) due to chronic renal failure (CRF), but the factors responsible are not known. The impact of this down-regulation on calcium sensitivity remains controversial. In vitro studies indicate decreased calcium sensitivity (elevated set-point) in parathyroid cells from CRF patients, but in vivo assessment of the set-point in CRF patients have yielded mixed results. Furthermore, in patients it is not possible to relate CaR levels to the four parameters of the calcium-PTH relationship. We have now observed a similar reduced CaR expression in hyperplastic PTGs of CRF rats. This model can be used to determine the role of decreased CaR in PTG hyperplasia and the abnormal calcium-PTH relationship, and to identify the factors that regulate the CaR. Definitive identification of the factors that directly control CaR levels requires an in vitro model with stable expression of the CaR. Traditional monolayer cultures do not respond to calcium. We have developed a unique parathyroid cell culture system that preserves the normal cellular architecture, allowing stable CaR expression and calcium response for several weeks. These cultures, which we refer to as pseudoglands or psGs, provide the best available model for studying the regulation off parathyroid cell CaR expression and provide a unique in vitro model t9o examine the effects in CaR levels on the calcium-PTH relationship. This grant presents an experimental approach that utilizes these in vivo and in vitro models to examine both the causes and effects of the down- regulation of the CaR in renal failure. The specific aims are: 1. To define the relationships, temporal and spatial, between parathyroid gland hyperplasia and down-regulation of the CaR in vivo. 2. To examine the effects of established therapies for uremic secondary hyperparathyroidism (phosphate restriction and vitamin D compounds) on CaR expression in uremic rats. 3. To determine the direct effects of potential regulators of CaR expression in vitro using the psG model. 4. To determine the effects of down-regulation of CaR on the calcium- PTH relationship and PTH expression in vivo and in vitro. These studies will provide new information about the effect of decreased CaR in CRF on the calcium-PTH relationship, define the factors responsible for the decrease, and test therapeutic strategies to correct the abnormality.

The production of lipid second messengers is stimulated by a variety of hormones and neurotransmitters that work through G protein coupled receptors (GPCR). One such lipid signaling system involves the hydrolysis of phosphatidylcholine, a major component of biological membranes, by phospholipase D (PLD) to produce phosphatidic acid (PA) and choline. Phosphatidic acid and its metabolic products, diacylglycerol and lyso-PA, have been postulated to modulate cell proliferation, vesicle transport membrane remodeling, long term goal of these studies is to achieve a better understanding of the molecular basis of PLD1 regulation. We decided to focus on the role of CDC42 in GPCR activation pathways because our preliminary findings suggests that Cdc42 modulates activation by other regulatory proteins and recent reports have shown that some heterotrimeric G proteins crosstalk to guanine nucleotide exchange factors specific to the Rho subfamily. The initial aim focuses on identifying sites on Cdc42 that bind and activate PLD1. We predict that several factors will be involved in heptahelical receptor activation of PLD1 and studies in the second aim will characterize two potentially novel modulators of PLD1 to determine their participation. The third aim will determine whether a purinergic receptor stimulation of PLD1 is transduced through Cdc42. The role of Cdc42 has not previously been systematically investigated. The proposed studies combine pharmacological, biochemical, and molecular genetic approaches to elucidate the molecular mechanisms of PLD1 activation in native cell lines. These studies will utilize both intact cells and cell-free preparations that retain purinergic receptor-stimulated activation of PLD1.

The objectives of Core H include: (1) a systems biology based identification of glycerophospholipids in primary macrophages, macrophage-like cell lines, and tissues; (2) expansion of methods for qualitative and quantitative analysis of lipids; (3) employing lipidomics to investigate macrophages and tissues under pathological conditions as disease models; and, (4) applying lipidomics to advance our understanding of the roles of lipids in metabolism. A systematic analysis continues in RAW264.7 and multiple types of primary macrophages to identify the components of the glycerophospholipid (GPL) lipidome. A variety of MS" and LC/MSn techniques identified over 850 GPL in RAW cells and we anticipate the identification of up to 1200 distinct species by the end of the second phase of this project. This tabulation includes the identification of a number of novel species (e.g., GPIns, GPA, and GPS ether-linked GPLs) and several atypical species not previously reported in macrophages. Core H developed a quantification system based on 20 odd-carbon diacyl internal standards from five GPLclasses and standard curves generated from dozens of even-carbon diacyl standards provided by the Lipid Synthesis Core. The planned addition of odd-carbon diacyl GPIns and six classes of lyso lipid internal standards, in addition to a wide variety of plasmanyl and plasmanyl-containing lipids, will greatly expand our ability to achieve absolute quantification of the vast numbers of chemically diverse phospholipid species. As described below, we also will participate in the Coordinated Studies proposed by this consortium that investigate the lipidomes of primary macrophages and tissues. In addition, a number of specialized studies will contribute to our understanding of the intersection of phospholipid metabolism with other classes of lipids and assist in the development of an integrated pathway map. We will take a leading role in defining the lipid molecular species produced during co-stimulation of TLR-4 with a variety of other inflammatory mediators (e.g., purinergic receptor subtypes), which will identify synergistic activations that are not observed upon stimulation with a single ligand. Such paradigms will provide a more physiological perspective of inflammatory pathways and identify new targets for pharmacological intervention in the treatment of diabetes, obesity, cardiovascular disease, cancer, and n neurodegeneration.

5,8,11,14,17-Eicosapentaenoic Acid; 5,8,11,14,17-Icosapentaenoic Acid; Aliquot; Alkynes; Analysis, Data; Animals; Antioxidants; Arts; Blood Plasma; Body Tissues; C element; Carbon; Cell Extracts; Cells; Class; Co element; Cobalt; Collaborations; Computer Analysis; Computer Programs; Computer Programs and Programming; Computer software; Data Analyses; Detection; Development; Diet; Dissociation; Eicosapentaenoic Acid; Fatty Acids; Glycerophospholipids; Goals; Heart; Housing; Human; Human, General; Infusion; Infusion procedures; Intermediary Metabolism; Investigators; LC/MS; Laboratories; Lipids; Liquid substance; Liver Extract; METBL; Mammals, Rodents; Man (Taxonomy); Man, Modern; Mass Spectrum; Mass Spectrum Analysis; Metabolic Processes; Metabolism; Method LOINC Axis 6; Methodology; Methods and Techniques; Methods, Other; Modeling; Molecular Weight; Monitor; Oxidative Stress; Pathway interactions; Pattern; Phosphatides; Phosphoglycerides; Phospholipids; Photometry/Spectrum Analysis, Mass; Plasma; Procedures; Programs (PT); Programs [Publication Type]; Proteins; Reaction; Research Personnel; Researchers; Reticuloendothelial System, Serum, Plasma; Rodent; Rodentia; Rodentias; Role; Sampling; Serum, Plasma; Software; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Sphingolipids; Standards; Standards of Weights and Measures; Systems Biology; Techniques; Time; Timnodonic Acid; Tissue Extracts; Tissues; Work; adduct; animal tissue; anti-oxidant; base; computational analysis; computer program; computer program/software; computer programming; design; designing; experiment; experimental research; experimental study; feeding; fluid; gene product; human study; liquid; liquid chromatography mass spectrometry; omega-3-Eicosapentaenoic Acid; pathway; peroxidation; programs; research study; social role